ZHANG ET AL. Biol Res 42, 2009, 121-132 121 Biol Res 42: 121-132, 2009 BR The possible role of light chain in myoblast proliferation

SU-ZHEN ZHANG1, A, YONG XU2, A, HUI-QI XIE3, A, XIU-QUN LI3, YU-QUAN WEI2, and ZHI-MING YANG3, *

1 Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School and School of Life Science, Sichuan University, Chengdu, 610041, P. R. China 2 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School and School of Life Science, Sichuan University, Chengdu, 610041, P. R. China 3 Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, 610041, P. R. China A These authors contributed equally to this work.

ABSTRACT

Skeletal muscles have the potential to regenerate by activation of quiescent satellite cells, however, the molecular signature that governs satellite cells during muscle regeneration is not well defined. Myosin light chains (Myls) are sarcomere-related as traditional regulator of muscle contraction. In this report, we studied the possible role of Myl in the proliferation of skeletal muscle-derived myoblasts. Compared to diaphragm-derived myoblasts, the extraocular muscle-derived myoblasts with lower levels of Myl proliferated faster, maintained a longer proliferation phase, and formed more final myotubes. It was found that blockading Myl with anti-Myl antibody or knockdown of Myl1 by siRNA targeted against Myl1 could enhance the myoblast proliferation and delay the differentiation of myoblasts. Our results suggested that Myl, likely Myl1, can negatively affect myoblast proliferation by facilitating myoblast withdrawal from cell cycle and differentiation.

Key terms: Myosin light chain, Proliferation, Myoblast, Extraocular muscle

INTRODUCTION far. Although compared to other skeletal muscles, the EOMs are inherently different Skeletal muscle possesses a highly in their molecular makeup and regenerative potential after severe injury or physiological properties, the efficient and a myopathic disease. However, in severity continuous regeneration and/or myogenesis of response to myopathic disease, skeletal in EOMs has been suggested as an muscle groups differ among themselves. important mechanism responsible for their For example, in Duchenne Muscular sparing in DMD (Fischer et al., 2002; Dystrophy (DMD), which is an X-linked McLoon et al., 2002; Fischer et al., 2005). recessive muscle-wasting disorder, Consequently, increasing the regenerative extraocular muscles (EOM) remain capacity of skeletal muscle could influence clinically spared, despite severe damage the evolution of congenital myopathies. seen in other skeletal muscles, especially in Muscle regeneration primarily relies on the diaphragm (Kaminski et al., 1992; the activation of a specific cell population Khurana et al., 1995; Porter et al., 2004). called satellite cells, which are normally The exact mechanisms remain unclear so quiescent and situated between the basal

* To whom correspondence should be addressed (Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Gaopeng Street, Keyuan Road 4, Chengdu, 610041, Sichuan, P. R. China. Tel.: (+86)28-85164088. Fax: (+86)28-85164088. E-mail: [email protected], [email protected] Received: March 28, 2008. In Revised form: January 2, 2009. Accepted: January 16, 2009 122 ZHANG ET AL. Biol Res 42, 2009, 121-132 lamina and the plasma membrane of cell proliferation. These findings, combined adjacent myofiber (Dhawan et al., 2005; Shi with our comparative proteomics study Dhawan et al., 2005). Upon activation, (data not shown), led us speculate that Myl satellite cells re-enter the cell cycle and may be involved in myogenesis or muscle proliferate. Ultimately, the progeny cells - regeneration through its function in myoblasts withdraw from the cell cycle and myogenic cells. Our study was undertaken fuse to form multinucleated myotubes to examine the problem by using myoblasts (Schultz et al., 1985; Conboy et al., 2003). derived from EOM and diaphragm muscles. The molecular signature that governs satellite cells during muscle regeneration is complex and incompletely understood. And MATERIALS AND METHODS the regulatory cascades await further study. In our comparative proteomics study Cell culture (data not shown), we identified myosin light chains (Myls) as the differentially Primary myoblasts were derived from EOM expressed proteins between EOM and the and diaphragm muscles of 3-wk-old diaphragm. Compared to the diaphragm, C57BL/6 mice using a modified preplate EOM expressed Myl1, Myl2 and Myl3 at technique (Qu-Petersen et al., 2002). Mice lower levels. Myls are the principal were euthanized by cervical dislocation, components in myofibrils and associated and EOM and diaphragm muscles were with myosin heavy chain heads. According isolated, digested with collagnase-type I to the conditions dissociated from the and dispase, and then centrifuged. The cells myosin heavy chains, Myls are divided into were first plated on 6-well plates for 2h two classes (Barton et al., 1985; Hailstones (preplate 1, pp1) in growth medium et al., 1990). One is called the regulatory (DMEM plus 20% FBS and 100 U/ml (or phosphorylatable) light chain (i.e. penicillin and 100 μg/ml streptomycin) at o Myl2) and the other is the alkali light chain 5% CO2 and 37 C. Then, the non-adherent (i.e. Myl1, Myl3 and Myl4). Each class has cells were transferred to other wells (pp2). several isoforms associated with different After 48h, the floating cells in pp2 were muscle types. For alkali Myls, Myl1, Myl3 collected and plated on new wells (pp3). and Myl4 are the fast-muscle isoform, After 48h, the pp3 cells were obtained. The slow-twitch / ventricular muscle isoform pp1 and pp2 cells were discarded because and embryonic / atrial muscle isoform, they contained non-myogenic cells (Qu et respectively. In vertebrate striated muscle, al., 1998; Qu-Petersen et al., 2002). The the acto-myosin interaction during muscle pp3 cells were expanded and passaged, and contraction is mainly regulated through the only passage 1 cells were used in the troponin/tropomyosin system, and Myls are following experiments. The purity and considered to have only a minor modulatory myogenic potential of the pp3 cells were effect on muscle contraction (Rushforth et evaluated by staining. Animals were al., 1998; Timson et al., 2003; Andruchov used in accordance with principles in the et al., 2006). Thus, the precise care and use of animals of the Sichuan physiological function of Myls in striated University. muscle is not as yet clear. Earlier studies showed that ventricular Myl2 has a role in Immunocytochemistry the regulation of cardiogenic development (Chen et al., 1998), which was supported by The myoblasts from EOM and diaphragm subsequent studies (Ghatpande et al., 2001). fixed with 4% paraformaldehyde on Jiang et al. (2002) found that precocious coverslips were permeabilized with 0.1% expression of MLC1f/3f (i.e. Myl1) results Triton X-100 in PBS at room temperature, in developmentally delayed mouse embryos then incubated with desmin antibody (1:100 and mesoderm ablation, and suggested that monoclonal mouse anti-desmin, Sigma) for 1 the precocious presence of muscle-specific h. After three rinses in PBS, the cultures contractile isoforms may disrupt were incubated with FITC-labeled secondary ZHANG ET AL. Biol Res 42, 2009, 121-132 123 antibodies and examined with a fluorescent used as the control. 48 hours following microscope (Nikon ECLIPSE 90i) equipped transfection, myoblasts were collected and with a digital camera (Nikon DXM 1200F). prepared for Western blot analysis with The nuclei were stained with DAPI. inmunoblotting. To further test the effect of Myl1 on Myoblast proliferation assay myoblast proliferation, EOM- and diaphragm-derived myoblasts were seeded Myoblasts from EOM and diaphragm at 7860 cells/cm2 in 96-well plates and were seeded at 4160 cells/cm2 in 96-well cultured overnight in the growth medium. plates, and cell number was determined After being transfected with Myl1-siRNA using the methylene blue assay (Oliver et al., (40 nM final), the cell number was 1989). Myoblasts were also seeded on the determined by methylene blue assay at 0, 1, coverslips at 7860 cells/cm2 to monitor cell 2, and 3d. proliferation, and cells in S phase were pulsed with 0.1 mM BrdU for 15 min at 1d, RT-PCR analysis 3d, and 5d, then fixed to detect BrdU- positive cells by immunocytochemistry as Total RNA was isolated from myoblasts at previously described (Knudsen et al., 1998). 24h after seeding using TRIzol reagents To demonstrate the effect of Myl on (Invitrogen). After synthesis of the first myoblast proliferation, diaphragm-derived strand cDNA, the following primers were myoblasts were seeded at 4160 cells/cm2 in used for specific PCR: for Myl1 96-well plates, and exposed to anti-Myl (product size 148 bp) were 5’- monoclonal antibody (Clone MY-21, TGCCCATGATGCAAGCTATCTC-3’ and Sigma) in increasing concentrations from 1 5’-ATCTTCTCTCCCAGAGTGGCGA-3’; to 1000 ng/ml. After 38h, cell proliferation for Myl2 (product size 258 bp) were 5’- was assessed as described above. The AGAACAGAGACGGCTTCATCG-3’ and proliferation assay showed that the 5’-TCAGCATCTCCCGGACATAGT-3’; maximum effect of anti-Myl antibody on for Myl3 (product size 197 bp) were 5’- myoblast growth was seen at 8 ng/ml when CCAAGAACAAGGACACTGGCA-3’ and the optical density tended to reach 5’-CGCTTCATAGTTGATGCAGCC-3’; maximum. Therefore, the following for Myl4 (product size 263 bp) were 5’- proliferation assay on myoblasts from EOM GCAAACCCAAGCCTGAAGAGA-3’ and and the diaphragm was performed in 5’-GTTGATGCAGCCATTGGCA-3’; and parallel with serial concentrations from 1 to for GAPDH (product size 191 bp) were 5’- 16 ng/ml. AACGACCCCTTCATTGAC-3’ and 5’- TCCACGACATACTCAGCAC-3’. PCR siRNA transfection of myoblasts products were resolved on 2% agarose gels. The band intensity ratio of the specific gene Inhibition of Myl1 expression in myoblasts to the internal standard (GAPDH) was used was achieved by RNA interference. Three to represent the level of gene expression. individual siRNAs against Myl1and nontargeted control siRNA (Ncontrol- Western blot analysis siRNA) were obtained from Ribobio (Guangzhou, China). The efficient silencing Whole-cell proteins from myoblasts at 24h of the Myl1 gene was assessed by after seeding and myoblasts at 48h after immunoblotting using a primary antibody Myl1-siRNA treatment were equally directed against Myl(Clone MY-21, resolved on SDS-PAGE in 12% acrylamide Sigma). Myl1-siRNA (40 nM final) or gels, respectively. The proteins were Ncontrol-siRNA (40 nM final) was transferred onto a PVDF membrane transfected into myoblasts using (Boehringer Mannheim GmbH, Germany) Lipofectamine 2000 according to the and probed with the Myl monoclonal mouse manufacturer’s instructions (Invitrogen). antibody (Clone MY-21, Sigma; 1:800). Lipofectamine 2000 without siRNA was Secondary HRP-conjugated anti-mouse IgG 124 ZHANG ET AL. Biol Res 42, 2009, 121-132 antibody (1:50000) were used in the cultures used in this experiment, we combination with ECL reaction (Rockford, immunostained the myoblast cultures with IL, Pierce). Densitometric analysis was desmin-specific antibodies. Figure 1 performed using the Quantity One 1-D showed that over 95% of primary cells analysis software (Bio-Rad). derived from EOM and diaphragm expressed desmin, indicating that the Myotube assay isolation procedure generated highly purified cultures of myogenic cells from Myoblasts from EOM and diaphragm both EOM and diaphragm. muscles were seeded in 6-well plates at EOM and diaphragm display different 7860 cells/cm2 and kept in growth medium; response to muscular dystrophy. Our the cells subsequently began to fuse into comparative proteomics studies (data not myotubes at low confluence (Qu-Petersen shown) have revealed that EOMs had a et al., 2002). The cultures were examined lower level of Myls (mainly Myl1, Myl2 every 2h until the emergence of myotubes, and Myl3) as compared with diaphragm and the final number of myotubes was muscles. To determine whether the counted from five random microscopic myoblasts from EOM and diaphragm have a fields and averaged at 144h after seeding. different expression of Myls, we harvested To assess the effect of Myl on myotube the EOM- and diaphragm-derived formation, diaphragm-derived myoblasts myoblasts at 24h after seeding, and the were seeded at 4160 cells/cm2 in 6-well expression of Myls in myoblasts was plates. Anti-Myl antibody was added at a measured by RT-PCR and Western blot dosage of 8 ng/ml, and the number of analysis. As seen in Figure 2A, the myotubes was determined at 16h and 144h expression levels of Myl1 and Myl4 mRNA respectively. To further determine the effect in EOM-derived myoblasts were both lower of Myl1 on myotube formation, diaphragm- than those in diaphragm-derived myoblasts. derived myoblasts were seeded at 4160 cells/ Neither Myl2 nor Myl3 mRNA was cm2 in 6-well plates and cultured overnight detected in these two myoblasts. Western (12h) in the growth media. Myl1-siRNA blot analysis showed that EOM-derived (40nM final) was then added to the cultures, myoblasts expressed Myl proteins at lower and the number of myotubes per well was level compared with diaphragm-derived counted at 48h after Myl1-siRNA treatment. myoblasts. These results indicated that Myl expressions in the EOM-derived myoblasts Statistical analysis were different from those in the diaphragm- derived myoblasts. Statistical analysis was performed using SPSS14.0. The significance between groups EOM-derived myoblasts proliferate faster was determined by ANOVA. All results are presented as means ± SD. The different regenerative potential of muscle groups may contribute to their heterogeneous response to muscular RESULTS dysthophy. Thus, we examined whether there is different proliferation potential The expression of Myls in proliferating between EOM-derived myoblasts and myoblasts derived from EOM and the diaphragm-derived myoblasts. We plated diaphragm myoblasts isolated from EOM and diaphragm muscles and determined their Previous works have demonstrated that proliferation using the methylene blue cell proliferating primary myoblasts express proliferation assay. Compared to the myoblast markers such as desmin, which is diaphragm-derived myoblasts, the EOM- expressed only in myogenic cells (Rando et derived myoblasts proliferated faster, which al, 1994; Qu-Petersen et al., 2002; resulted in a significant increase in the McCroskery et al., 2003). To characterize number of cells (Fig. 2B). We also ZHANG ET AL. Biol Res 42, 2009, 121-132 125 monitored the myoblast proliferation by expressed Myls at relatively high levels. pulsing myoblasts in the S phase of the cell These findings lead us to speculate that cycle with BrdU at specific time points. there could be a relationship between Myl Cells labeled with BrdU reflect that they and the proliferative capacity of muscle are in a proliferating state. Figure 2C cells. Thus, we tested the function of Myl in showed that the percentages of BrdU- myoblast proliferation. For this purpose, an positive cells in EOM-derived myoblasts antibody specific for Myl was added to the were significantly higher than those in myoblast cultures in a series of diaphragm-derived myoblasts at various concentrations (from 1 to 1000 ng/ml). time points, suggesting that more EOM- Because there is a higher level of Myl in derived myoblasts were in proliferating diaphragm-derived myoblasts, we thought state when compared with the diaphragm- that it should be more sensitive to antibody. derived myoblasts. Consequently, we carried out the examination in diaphragm-derived Myl blockade or knockdown increases the myoblasts first. As shown in Figure 3A, we myoblast proliferation observed significant increases in cell proliferation at 38h after antibody Proliferation assays clearly demonstrated treatment, they displayed dose-dependence that EOM-derived myoblasts with lower on antibodies in a given range. When the levels of Myls proliferated faster than concentration reached 8 ng/ml, the effect of diaphragm-derived myoblasts that the antibody tended to peak. These results

EOM Diaphragm Negative

Fig. 1: Expression of desmin in EOM- and diaphragm-derived myoblasts. Primary myoblasts were cultured from the EOM and diaphragm muscles, fixed, and immunostained for desmin. The nuclei stained with DAPI in the corresponding fields were shown; Immunostaining without primary antibody was used as negative control. Greater than 95% of the primary cultured cells were myogenic. Bar, 10μm. 126 ZHANG ET AL. Biol Res 42, 2009, 121-132 showed that the anti-Myl antibody exerted a and anti-Myl antibody was added in neutralizing role. Based on these results, a increasing concentrations. Thirty-eight series of concentrations, ranging from 1 to hours after treatment with the anti-Myl 16 ng/ml, were adopted by subsequent antibody, the proliferation rate of experiments. The myoblasts isolated from myoblasts from EOM and disphragm were both EOM and the disphragm were cultured enhanced (Fig. 3B).

A

B C

Fig. 2: EOM-derived myoblasts with lower level of Myl proliferate faster. (A) Expression of Myls in myoblasts. Myl1-4 mRNA expressions were analyzed by RT-PCR and Myl protein was analyzed by Western blot. Myl2 and Myl3 amplicons were not detected at the proliferateive myoblasts. The results are expressed as means ± SD from three independent experiments. *p < 0.05, vs EOM. (B) Proliferation curves. EOM- and diaphragm-derived myoblasts proliferation rate was determined by the methylene blue assay. Experiments were performed in quadruplicate, and three independent experiments were done. *P<0.01 vs diaphragm. (C) Proliferation assay by BrdU labeling. Myoblasts in S phase were pulsed with BrdU for 15 min at 1d, 3d, and 5d, then fixed and immunostained for BrdU. Data are from three independent experiments, with at least 500 cells scored per experiment. *P<0.05 vs EOM. ZHANG ET AL. Biol Res 42, 2009, 121-132 127

We also tested the effect of Myl (mainly were efficiently suppressed by Myl1-siRNA Myl1) on myoblast proliferation by using and the proliferation of these cells was specifically-targeted siRNA to delete the significantly enhanced after treatment with expression of Myl1 in myoblasts. Figure 3C Myl1-siRNA. These results indicated that and D showed that the Myl1 expressions in Myl, likely Myl1, may negatively affect EOM- and disphragm-derived myoblasts myoblast proliferation.

A B

D

C

Fig. 3: Increase of myoblast proliferation by Myl blockade and deletion. (A) Diaphragm-derived myoblasts were incubated with anti-Myl antibody in increasing concentrations from 0 to 1000ng/ ml. By 38h, the proliferative potential of the myoblasts increased in a dose-dependent manner and tended to peak at the concentration of 8ng/ml. (B) EOM- and diaphragm-derived myoblasts were cultured in the medium containing anti-Myl antibody from 1 to 16ng/ml for 38h, and PBS was used as the control. The assays were performed in quadruplicate and means ± SD from three independent experiments. *P < 0.01 (EOM, as compared with control); #P < 0.01 (diaphragm, as compared with control). (C) Myoblasts from EOM and diaphragm were transfected with Myl1-siRNA (40 nM final) or Ncontrol-siRNA (40 nM final) using Lipofectamine 2000. The vehicle without siRNA was used as the control. After 48h, Myl expressions in myoblasts were detected by Western blot analysis (1: Control; 2: Ncontrol; 3: Myl1-siRNA). (D) EOM- and diaphragm-derived myoblasts were transfected with Myl1-siRNA (40 nM final), then cell counting was made by the methylene blue assay at the indicated time points. The assays were performed in quadruplicate and the data were expressed as mean ± SD from three independent experiments. *P < 0.05, vs Control; #p <0.05, vs Ncontrol. 128 ZHANG ET AL. Biol Res 42, 2009, 121-132

Delayed differentiation potential of but increased at 144h, revealing that Myl myoblasts with lower level of Myl or blockage delayed differentiation of blockage of Myl myoblasts. Figure 5B also showed significant delay in myotube formation after Myogenic differentiation of myoblasts Myl1-siRNA treatment. These results occurs based on withdrawal of actively demonstrated that Myl, likely Myl1, growing myoblasts from the cell cycle into significantly affected the process of the G0 phase. Myoblasts cultured in growth myoblast proliferation by facilitating the medium could differentiate and fuse into differentiation potential of myoblasts. myotubes at low confluence (Qu-Petersen et al., 2002). Thus, we performed myotube experiments to investigate the differentiation DISCUSSION potential of myoblasts from EOM and diaphragm. Diaphragm-derived myoblasts The present study was designed to test the began to yield myotubes at 16h after possible role of Myl in myoblast seeding. But myotubes from EOM-derived proliferation. Compared to diaphragm- myoblasts did not emerge until 40h after derived myoblasts that displayed a higher seeding (Fig. 4). At 144h, however, the expression of Myl combined with a slow number of myotubes derived from EOM was proliferation and a rapid fusion into significantly higher than that of myotubes myotubes, EOM-derived myoblasts from diaphragm myoblasts. displayed a lower expression of Myl, To determine whether Myl affects the combined with a faster proliferation and a differentiation potential of myoblasts, we delayed fusion into myotubes. Upon treated myoblast cultures with anti-Myl blockade of Myl or knockdown of Myl1 antibody and Myl1-siRNA and observed in myoblasts, the proliferation rate of myotube formation. Because diaphragm myoblasts was enhanced and the myotube myotubes formed faster, only diaphragm- formation was delayed. Our results derived myoblasts were used in the suggested that Myl likely negatively experiment. As shown in Figure 5A, after affect the myoblast proliferation by anti-Myl antibody treatment, the number of facilitating the terminal differentiation of myotubes at 16h significantly decreased, myoblasts.

Fig. 4: Myotube formation of different myoblasts. Myoblasts derived from EOM and diaphragm muscles were cultured in growth medium. At 16 h after seeding, diaphragm-derived myotubes appeared. But EOM derived myoblasts did not form myotubes until another 24 h. At 144 h, myotubes were counted from five random microscopic fields (200×) and averaged. The assays were performed in duplicate and means ± SD from three independent experiments. *P < 0.01 vs EOM. Bar, 50μm. ZHANG ET AL. Biol Res 42, 2009, 121-132 129

The Myls isoform profile, in developing et al., 1993; Sutherland et al., 1990). Here, muscle cells in vivo or in cultured muscle our results of undetectable Myl2 and Myl3 progenitor cells in vitro, has been mRNA in cultured myoblasts were extensively explored in a variety of species concurrent with these findings. Myl1 and (Keller et al., 1980; Wade et al., 1989; Myl4 gene expression is induced during Sutherland et al., 1993; Faerman et al., myogenesis (Keller et al., 1980; Sutherland 1993). It is generally considered that Myls, et al., 1993). However, some studies as well as other muscle specific contractile showed that in earlier development embryo, proteins, are synthesized at the onset of Myl4 and Myl1 and fast-muscle Myl2 myoblast differentiation. However, (Mylpf) are sequentially expressed in different Myl isoforms display a undifferentiated myoblasts (Faerman et al., heterogeneous pattern of expression. Myl3 1993; Lyons et al., 1990). Using is not expressed during myogenesis in comparative protoemic strategies, Tannu et culture and can be detected only in mature al. (2004) also found that proliferating skeletal muscles (Wade et al., 1989; Cohen- C2C12 myoblasts express Myl1 protein. Haguenauer et al., 1989), as well as the These results indicated that Myl1, Mylpf slow-muscle isoform of Myl2 (Sutherland and Myl4 gene expression could be

Fig. 5: Effects of Myl blockade and knockdown on differentiation of myoblasts. (A) Diaphragm- derived myoblasts were treated with anti-Myl antibody (8ng/ml). Afterwards, the number of myotubes per well was quantified at 16h or was calculated from five random microscopic fields (×200) and averaged at 144h. The assays were made in triplicate and means ± SD from three independent experiments. *P<0.01 vs Control. Bar, 50μm. (B) Diaphragm-derived myoblasts cultured overnight (12h) were treated with 40nM Myl1-siRNA. The number of myotubes was counted per well at 48 h after treatment. The assays were made in triplicate and means ± SD from three independent experiments. *P<0.01 vs Control and Ncontrol. Bar, 50μm. 130 ZHANG ET AL. Biol Res 42, 2009, 121-132 activated before the onset of myogenesis. In Aβ in cultured amyloid precursor protein our study, at 24h after seeding, Myl1 and mutant neurons or primary neurons Myl4 mRNA in EOM- and diaphragm- (Tampellini. et al, 2007). However, the derived myoblasts were both detected, as biological mechanisms by which the well as Myl protein. It suggested that endocytosed antibody comes in contact myoblasts could express Myl prior to with its target antigen remains unclear. In differentiation, because at this time most the present study, when the anti-Myl myoblasts were in the proliferating state. antibody concentration was high, its effect We found that EOM-derived myoblasts on cell proliferation was lost. This may be had a lower level of Myls compared with due to the cytotoxic effect of the antibody diaphragm-derived myoblasts. However, or the antibody preservation solution. the EOM-derived myoblasts proliferated Upon activation, more quiescent faster than the diaphragm-derived myoblasts re-enter the S phase from G0/G1 myoblasts, which is consistent with to undergo cell cycle progression leading to previous studies (McLoon et al., 2002). the increased proliferation rate of myoblasts Studies by Jiang et al. (2002) suggested (McCroskery et al., 2003). Alternatively, that precocious expression of MLC1f/3f the increase of myoblast proliferation may (i.e. Myl1) could disrupt cell proliferation be due to the delayed withdrawal of in developmental mouse embryos. growing myoblasts from the cell cycle. According to these results, we therefore After withdrawal from the cell cycle, proposed that the level of Myl expression myoblasts differentiate into myotubes. The could be related to myoblast proliferation. later the myoblasts withdraw from the cell In accordance with the hypothesis, the cycle, the more the myotubes might be proliferation of myoblasts was enhanced formed due to a longer proliferating phase. with the neutralization of Myl by anti-Myl Our myotube experiments confirmed this antibody or deletion of Myl1 by RNA prediction. Tannu et al. (2004) found that interference, suggesting that a low level of the proliferating C2C12 myoblasts had a Myl in myoblasts likely facilitated the lower expression level of Myl1 than their myoblasts proliferation. Thus, Myl, likely differentiated myotubes. Works by Myl1, may play a negative role in Kislinger et al. (2005) revealed that the myoblast proliferation. There are expression of Myl1 in differentiating accumulating data pointing to the myotubes from C2C12 myoblasts increased effectiveness of protein blockage by a progressively following induction of the specific antibody (Ma et al., 1991; Sun et differentiation program. In this study, we al., 2002; Schilling et al., 2003; Tampellini found that the blockage of Myl with anti- et al., 2007). As an extracellular molecule, Myl antibody or deletion of Myl1 by RNA antibodies can enter cells in culture interference delayed the differentiation of through membrane receptor-dependent myoblasts. These results suggested that a endocytosis and then exert their function. lower level of Myl may delay the For example, antibodies to a ribosomal withdrawal of myoblasts from the cell cycle protein have been shown to penetrate cells and terminal differentiation, consequently in culture through binding to the P0 increasing the potential for the myoblast protein on cell surfaces and causing proliferation. It is also noteworthy that the profound inhibition of protein synthesis BC3H1 muscle cell line, which lacks MyoD (Koscec et al, 1997) or apoptosis of cells (the member of the myogenic regulatory (Sun et al, 2001). Studies by Schilling et al gene family), does not express Myl1 and (2003) reported that endocytosed anti-HBs also does not form multinucleate myotubes antibody, which was mediated by FcRn- during differentiation (Brennan et al., receptor, inhibited the secretion of HBV 1990). Although it was thought that the virions, and in particular HBsAg, from failure to express MyoD was responsible HBV infected hepatocyte lines. Recent for the defect in differentiation, it was also studies showed that internalized Aβ concluded that Myl1were potentially antibodies decrease levels of intracellular functional in BC3H1 cells. ZHANG ET AL. Biol Res 42, 2009, 121-132 131

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